There is an interest in surface sensitive techniques for quantifying molecular interactions. Properties that are of interest are e.g. concentration of free analyte in solution, surface concentration of molecules on sensor surface, reaction kinetics between interacting substances, affinity of said substances, allosteric effects or epitope mappings. Examples of interacting substances are antigen-antibody, protein-protein, receptor-ligand, DNA-DNA, DNA-RNA, peptides-proteins, carbohydrates-proteins, glycoproteins-proteins, etc.
There are many techniques that are suitable for this task, e.g. surface plasmon resonance (SPR), resonant mirror, grating couplers, interferometers, surface acoustic wave (SAW), Quartz Crystal Microbalance (QCM) etc. So far, SPR is the dominating technique.
Areas of application are e.g. measurement of concentration of substances in biological research, biochemistry research, chemical research, clinical diagnosis, food diagnostics, environmental measurements, etc. Kinetic measurements can be used to determine rate constants as kon and koff. Affinity measurements can be used to determine equilibrium association (KA) or dissociation (KD) constant as well as avidity.
SPR is a well-known phenomenon that consists of a bond electromagnetic wave, due to oscillations of electrons at the interface of a plasma. The surface plasmon can only exist at an interface between said plasma (e.g. a metal) and a dielectricum. A change in the optical constants of the dielectricum will change the propagation constant of the surface plasmon. The surface plasmon can be excited by light if the propagation constant of the light parallel to the interface is equal to, or close to, the propagation constant of the surface plasmon. Normally one uses the Kretschmann configuration [1] where a thin metallic film is applied on a prism, having a higher refractive index than the measured sample. The surface plasmon is then evanescently excited under total internal reflection, i.e. at an incident angle, normal to the surface, larger than the critical angle. At a certain incident angle, the component of the wave vector parallel to the surface meets the real part of the complex wave vector for a surface plasmon, and hence the light will couple into the surface plasmon and propagate at the interface between said plasma and said dielectricum. The surface plasmon will reradiate into the prism, and for a certain thickness of said plasma a destructive interference occur, leading to zero or close to zero intensity of reflected light. For a smooth surface of said plasma, coupled light will be absorbed in said plasma and generate heat.
When molecules bind close to the interface (within the probe depth of the surface plasmon) the interaction can be detected by a shift in the resonance condition of the surface plasmon. This can be detected as a shift in a reflected light intensity.
The SPR sensor can be used in an imaging mode, also denoted microscopy. This was at first proposed by Yeatman in 1987 [2]. Other setups are proposed by Bengt Ivarsson EP958494A1: ANALYTICAL METHOD AND APPARATUS [3, 4], or GWC Instruments SPRimager [5]. The latter utilizes many wavelengths in a non-simultaneous manner.
The surface plasmon resonance (SPR) phenomenon was already described in 1959 [6] and SPR apparatuses for thin adlayer analysis have been thoroughly described since 1968 [1, 7]. SPR setups for biosensing were used for the first time in 1983 [8] and for imaging applications in 1987 [2, 9]. With imaging SPR, also denoted SPR microscopy, new applications arise, e.g., label free—real time—multi spot biochemical analyses [10, 11], which can increase the throughput tremendously. The pioneering work on imaging SPR was undertaken by Knoll et al., who investigated surfaces patterned with Langmuir-Blodgett films [12, 13]. They also investigated the physical aspects of the technique, including lateral resolution [14], and proposed different setups, e.g. the rotating grating coupler [15].
There are in principal three different ways to measure changes in the SPR propagation constant. First, by measuring the reflected intensity (reflectance) at a flank of the SPR dip at a certain wavelength and incident angle. Second, by measuring the intensity of the reflected light versus the angle of incident light (angular interrogation). Third, by measuring the intensity of reflected light for different wavelengths at a certain incident angle (wavelength interrogation).
For zero-dimensional SPR (measurement of a single spot) said angular or wavelength interrogation requires at least a one-dimensional (linear) detector to make an instant measurement of the position of an SPR dip. For one-dimensional SPR (measurement of a single line) said angular or wavelength interrogation requires at least a two-dimensional (matrix) detector to make an instant measurement of the position of an SPR dip. In this case one dimension is used for the length scale (real image) and one dimension is used for the dip (either angle or wavelength). If two-dimensional SPR-measurement is performed, normally a dip cannot be resolved, i.e. one can normally only make an intensity measurement with a two-dimensional detector, i.e. for the two length scales. This means that only a limited portion of the dynamic range (effective refractive index of the sample) can be measured, due to the limited extension of the SPR-dips (in either angel or wavelength). Only at a small range will the slope of the SPR-dip be high, which means that there will be a limited range of high sensitivity.
To overcome these drawbacks the present invention provides a two-dimensional imaging surface plasmon resonance apparatus wherein a set of wavelengths can simultaneously (or pseudo-simultaneously) be used, e.g. by using a multi-wavelength light source and a color camera.